William Halpern

Mechanical behavior of pressurized in vitro prearteriolar vessels determined with a video system

The muscular resistance arteries of the mesentery and brain serve two different control functions in the cardiovascular system. The former are representative vessels of vascular beds that influence total peripheral resistance and blood pressure; the latter are a good model of vessels in beds that demonstrate blood flow autoregulation. Our purpose was to develop a versatile myographic system appropriate for the in vitro study of 75–250 μm diameter vessels and to explore different physiological properties of cerebral and mesenteric arteries. In this paper the system is described in detail, examples of its use in determining the dynamic responses of the vessels to electrical stimulation are provided, and certain measures indicative of the extent of myogenic behavior are characterized.Cylindrical artery segments about 3-mm long were dissected from Wistar-Kyoto rats and mounted in a chamber filled with physiological saline solution maintained at 37°C. The same solution was perfused via a syringe into one end of the vessel through a microcannula. The other end was then occluded so that experiments could be made over a wide range of transmural pressures without flow. The vessel was viewed through a microscope coupled with a TV camera, and the video output signal of a selected scan line was processed by an electronic dimension analyzing system. This permitted simultaneous digital presentation and analog voltage outputs of the vessel wall thicknesses and lumen diameter. We further incorporated servo control of the syringe using a motor drive. In this way, vessel tests could be carried out at constant pressure or constant diameter, and vessel responses could be obtained following either pressure or diameter command signals.Using the methods presented in this study, small vessels can be maintained under conditions that approximate their in vivo state more closely than other in vitro techniques using ring segments on wires. We also find that the opto-electronic instrumentation is ideally suited for studying the dynamic vessel properties that underlie the control of vascular smooth muscle.

Mechanical and morphological properties of arterial resistance vessels in young and old spontaneously hypertensive rats.

We studied alterations in structural and mechanical properties of mesenteric arterial resistance vessels from young (6-week) and old (50-week) spontaneously hypertensive (SHR) and matched normotensive Wistar-Kyoto (WKY) rats. Emphasis was placed upon relating the active tension capabilities of these vessels to their smooth muscle cell content. Cylindrical segments, 0.7 mm long with internal diameters of 150 μm, were mounted in a myograph capable of recording circumferential vessel wall tension and dimensions. Comparisons of vessel morphology and mechanics were performed at a normalized internal circumference, L1, where active tension (AT1) is near maximum. Arterial wall and medial hypertrophy were observed in young and old SHR. Since the percent smooth muscle cells within the media for SHR was similar to that of WKY, both increased smooth muscle cell and connective tissue content account for the medial hypertrophy. These differences in SHR vessels were reflected directly in their passive and active mechanical properties. Fully relaxed vessels from SHR were less compliant, and upon activation at Li (high potassium depolarization), AT, was not different for young SHR and WKY, but values for old SHR were 35% greater (P < 0.05) than for WKY. When relating the active force generation of the vessel to the actual smooth muscle cell area, values for smooth muscle cell stress (force/area) were similar for SHR and WKY at both ages. In addition, similarities were observed for active dynamic mechanical measurements of Youngs modulus and half response time. Genetic hypertension in rats therefore appears to be associated with the development of increased vessel contractility determined by a greater number of smooth muscle cells which possess contractile properties similar to those of normotensive vessels. Ore Res 45: 250-259, 1979

Norepinephrine Sensitivity, Tension Development and Neuronal Uptake in Resistance Arteries from Spontaneously Hypertensive and Normotensive Rats

Intact segments of mesenteric resistance arteries (200 micron) from 5-month-old spontaneously hypertensive (SHR) and normotensive Wistar Kyoto (WKY) rats were tested for norepinephrine (NE) sensitivity. Dose-response curves were obtained both before and after adrenergic denervation produced by short-term, in vitro, 6-hydroxydopamine (6-OHDA) treatment. NE sensitivities of innervated vessels were the same in SHR and WKY rats, however, after 6-OHDA, not only did both ED50s show significant decreases (8.7- and 3.8-fold, respectively), but the ED50 of SHR vessels was half that of WKY (NE sensitivity increased twofold). In addition there was a 33% increase in wall tension generated in response to maximum NE stimulation, and a 44% increase in neuronal NE uptake in the SHR vessels. These results show that several important alterations have occurred in resistance vessels from SHR rats in the established phase of hypertension. The relationship of the increase in neuronal uptake to the increased total peripheral resistance and increased vascular reactivity commonly seen in SHR perfused beds and whole animals is unknown. However, the increase in NE sensitivity and in maximum NE wall tension could contribute to increase in both of these characteristics.

Impaired potassium-induced dilation in hypertensive rat cerebral arteries does not reflect altered Na+,K(+)-ATPase dilation.

We have recently demonstrated that K(+)-induced dilation of cerebral resistance-sized vessels has two independent components, only one of which seemed sodium pump dependent. In our current investigation, potassium-induced dilation of spontaneous tone was compared in cerebral arteries from normotensive Wistar-Kyoto rats and age-matched stroke-prone spontaneously hypertensive rats. Branches of the posterior cerebral artery were cannulated and pressurized, and these vessels developed spontaneous tone. After a 5-minute period in K(+)-free physiological saline solution, K+ was increased in 1-mM increments to a final concentration of 15 mM. In the normotensive arteries, K+ concentrations between 0 and 5 mM K+ resulted in dilations that had a transient (sodium pump-dependent) component, and K+ concentrations in excess of 7 mM produced dilations that lacked a transient (sodium pump-independent) component. Similar branches from the hypertensive rat also responded with transient dilations to K+ (less than 5 mM), and these were significantly greater at 3 mM K+. However, the maintained dilations to K+ (greater than 7 mM), noted in preparations from Wistar-Kyoto rats, were absent in seven of eight preparations. Thus, the impaired dilations, in the hypertensive vessels, to K+ described here is a consequence of altered function of some sodium pump-independent component rather than altered Na+,K(+)-ATPase activity.

In vitro Methodology for Resistance Arteries

Two commonly used methods for examining the physiological and pharmacological properties of isolated resistance arteries are the ring-mounted preparation and the cannulated, pressurized vessel. Each technique is discussed and consideration given to limitations and advantages. Also presented are examples of comparative differences between them, and practical experimental schemes for calibrating cannulae and for perfusing resistance arteries. Although both methods are valuable, the cannulated approach may better reflect the in vivo properties of the arteries.

Altered Membrane Electrical Properties of Smooth Muscle Cells from Small Cerebral Arteries of Hypertensive Rats

Certain electrophysiological properties of smooth muscle cells within middle cerebral arteries of spontaneously hypertensive rats (SHR) and their normotensive Wistar-Kyoto (WKY) controls were recorded with glass microelectrodes. Under control conditions the smooth muscle cells of arteries from SHR rats exhibited fast and slow oscillations of the resting membrane potential (Em). In marked contrast the smooth muscle cells of WKY cerebral arteries were electrically quiescent. The Em vs. log [K]o relationship was similar for arteries from both SHR and WKY rats and extrapolated to similar [K]i values of 150-155 microM when determined under control conditions. When Em vs. log [K]o curves were obtained in the presence of ouabain to block electrogenic Na-K transport, the smooth muscle cells of SHR cerebral arteries exhibited a lower mean slope per decade change in [K]o (i.e., depolarized more at various concentrations of [K]o in the presence of ouabain) when compared to WKY. Arterial smooth muscle cells from SHR also depolarized to a greater extent when exposed to zero K+ solutions. The results of this study indicate that smooth muscle cells of cerebral arteries from SHR rats have a greater electrogenic Em component as well as altered ionic conductances for K+ and possibly Na+ both of which may contribute to their spontaneous electrical activity.

Reactivity of isolated porcine coronary resistance arteries to cholinergic and adrenergic drugs and transmural pressure changes.

The reactivity of porcine intramyocardial resistance arteries (223 ± 7 μm i.d., n = 30) was investigated with a pressurized in vitro preparation. Diameter changes in response to acetylcholine and to adrenergic drugs and dynamic changes in transmural pressure changes were investigated. Acetylcholine produced concentration-dependent constrictions, causing maximal reductions of 71 ± 3% in lumen diameter, with EC50 values averaging 1.9 ± 10-7 M (n = 7). These responses were inhibited by atropine (10-7 M) and therefore were mediated by muscarinic receptors. In addition, acetylcholine did not elicit relaxation in nine out of 10 vessels precontracted with U46619 (10-7 M). Norepinephrine and epinephrine never produced constrictions (n = 6) even in the presence of propranolol (10-6 M). Both norepinephrine and isoproterenol caused dose-dependent relaxations in acetylcholine-precontracted vessels, with IC50 values of 8.2 ± 10-7 M (n = 5) and 6.6 ± 10-2 M (n = 6), respectively. These relaxations were suppressed by propranolol. Between transmural pressures of 10 and 90 mm Hg, there was no intrinsic myogenic tone (n = 7). In addition, the vessels responded only passively to sudden pressure changes of 40 mm Hg. In all vessels, the functional integrity of the endothelium was verified by relaxations to substance P (10-8 M) and/or bradykinin (10-2 M). This is the first in vitro study of coronary resistance arteries that demonstrates that 1) acetylcholine is a potent constrictor of these arteries, suggesting that parasympathetic mechanisms may play an important role in coronary blood flow regulation; 2) α-adrenoreceptor influence is minimal or entirely absent; and 3) these arteries do not possess an intrinsic myogenic tone and respond passively to transmural pressure change. Hence, myogenic mechanisms do not appear to be of primary importance in the autoregulation of coronary blood flow.

Effects of Antihypertensive Drug Therapy on the Morphology and Mechanics of Resistance Arteries from Spontaneously Hypertensive Rats

Spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto rats (WKY), 25 weeks of age, were treated for 23 weeks with hydrochlorothiazide, hydralazine, and reserpine added to their drinking water. Cylindrical segments of mesenteric arteries (about 170 micrometers diameter, and 0.7 mm long) were isolated and mounted in a myograph for dimensional and circumferential tension measurements. Treatment of the SHR and WKY decreased systolic blood pressures by 29% and the smooth muscle cell (SMC) content of these vessels by 31%. A linear relation was found between blood pressure and SMC content for all treated and untreated rats. Our results suggest that effect of treatment on the cellular content of the media was sufficient to account entirely for the maintenance of the lowered blood pressure.

Differential Contractile Responses of Pressurized Porcine Coronary Resistance-Sized and Conductance Coronary Arteries to Acetylcholine, Histamine and Prostaglandin F2α

The reactivities of porcine resistance-sized arteries (270 microns o.d.) and conductance coronary arteries (4.2 mm o.d.) were compared in the presence of a functional and morphologically intact endothelium using a pressurized in vitro preparation. In resistance vessels, acetylcholine (ACh) produced maximal diameter reductions of 42% and the EC50 was 0.19 microM. Constrictions to histamine and prostaglandin F2 alpha (PGF2 alpha), however, were either absent or minimal. In contrast, maximum diameter constrictions of conductance vessels were 19, 33 and 22% in response to ACh, histamine and PGF2 alpha, respectively. Corresponding EC50 values were 0.58, 2.8 and 2.9 microM. The significant differences in reactivity between resistance and conductance arteries underscore the potential importance of regional specialization in the coronary blood flow regulation.

Asymmetry of responses to norepinephrine in perfused resistance arteries

Diameter constrictor responses were significantly greater when norepinephrine (NE) was delivered intraluminaly compared to extraluminal application in isolated perfused mesenteric resistance arteries of the rat. The difference in response via the two routes was abolished by cocaine, a neuronal uptake blocker. Endothelium removal enhanced NE-induced constrictions but responses remained significantly greater when delivered intraluminally. This study provides evidence of asymmetry of vascular smooth muscle in resistance arteries due to a greater neuronal uptake of NE on the adventitial surface.